The ebs2 mutation partially suppresses the bri1–9 mutant. Images of 7-d-old bri1–9, WT, and ebs2–1 bri1–9 grown on half-strength Murashige and Skoog medium(A); 5-week-old soil-grown plants of bri1–9, WT, and ebs2–1 bri1–9 (B); 7-week-old mature plants of bri1–9, WT, and ebs2–1 bri1–9 grown in soil (C); and 4-d-old dark-grown seedlings of bri1–9, WT, and ebs2–1 bri1–9 (D). (E) Average hypocotyl length of 4-d-old etiolated seedlings of bri1–9, WT, and ebs2–1 bri1–9. Each bar represents the average of more than 40 seedlings of duplicated experiments. Error bar denotes SE.

CRT3 is the major lectin that retains bri1–9 in the ER. (A) CRT3 interacts with bri1–9 in an UGGT-dependent manner. (B) The CRT3-bri1–9 interaction is abolished in ebs2–1. For A and B, anti-GFP immunoprecipitates from 18-d-old whole seedlings of WT, or transgenic lines were separated by 10% SDS/PAGE and analyzed with anti-GFP and anti-CRT3 antibodies. IgG heavy-chain interference is indicated. (C) Shown here are 18-d-old seedlings of bri1–9, crt1 bri1–9, crt2 bri1–9, crt1 crt2 bri1–9, ebs2–8 bri1–9, and WT grown on half-strength Murashige and Skoog medium. (D) Shown here are 18-d-old seedlings of bri1–9, cnx1 bri1–9, cnx2 bri1–9, cnx1 cnx2 bri1–9, and WT grown on half-strength Murashige and Skoog medium. (E) Three-week-old transgenic ebs2–8 bri1–9 seedlings containing pPZP222 vector, a gCNX1 transgene, or a gCRT3-cCRT3 transgene. (F) Shown here are 6-week-old soil-grown bri1–9 and 4 gCRT3-cCRT3 ebs2–8 bri1–9 transgenic plants. (G) Analysis of CRT3 protein abundance in gCRT3-cCRT3 ebs2–8 bri1–9 transgenic lines shown in F. Protein extracts of 3-week-old transgenic plants were separated by 10% SDS/PAGE and analyzed by immunoblotting with an anti-CRT3 antibody. After stripping off bound IgGs, the filter was re-probed with anti-BiP antibody. The numbers indicate molecular weight (in kDa), triangle represents CRT3, and star denotes BiP for loading control. (H) The C terminus is crucial for the “retainer” function of CRT3. Shown here are 6-week-old soil-grown transgenic ebs2–8 bri1–9 mutants expressing the pCRT1:gCRT1N-CRT3C or pCRT3:gCRT3N-CRT1C chimeric transgene.

EBS2 functions in quality control of bri1–9. Images of 6-week-old soil-grown det2 and ebs2–2 det2 mutants (A); 4-week-old soil-grown plants of bri1–101 and ebs2–2 bri1–101 (B); 5-week-old soil-grown bri1–5 and ebs2–2 bri1–5 mutants (C); and 5-week-old soil grown bri1–6 and ebs2–1 bri1–6 mutants (D). (E) Quantitative analysis of BR sensitivity. Root lengths of 7-d-old seedlings grown on BL-containing medium were measured and presented as the relative value of the average root length of BL-treated seedlings to that of mock-treated seedlings of the same genotype. Each data point represents the average of approximately 30 seedlings of duplicated experiments. Error bar denotes SE. (F) BR treatment leads to BES1 dephosphorylation: 2-week-old seedlings were treated with 1 μM BL for 1 h. Total proteins were extracted in 2× SDS buffer, separated by 10% SDS/PAGE, and analyzed by immunoblotting using anti-BES1 antibody (24). Coomassie blue staining of RbcS served as a loading control. (G) Endo H sensitivity of BRI1 and bri1–9. Equal amounts of proteins of 4-week-old leaves from different samples were treated with 1 μL of Endo Hf for 1 h at 37°, followed by Western blot analysis with anti-BRI1 antibody. (H) Confocal examination of subcellular localization of bri1–9:GFP and BRI1:GFP in root tips of 6-d-old light-grown seedlings.

Molecular cloning of EBS2. (A) EBS2 was mapped to a 150-kb genomic region between markers T23G18_2 and F22O13_1 on the top of chromosome I. The line represents genomic DNA, and markers and numbers of recombinants are shown above and below the line, respectively. (B) The gene structure of EBS2. EBS2 contains 14 exons (black bar) and 13 introns (line). Gray boxes denote untranslated regions, lines indicate positions of ebs2 mutations, and triangle denotes T-DNA insertion. (C) Nucleotide change and molecular defects of 8 ebs2 alleles. (D) Alignment of CRT sequences from Arabidopsis thaliana (At), Oryza sativa (Os), and Homo sapiens (Hs). Comparison of AtCRT3 (NP_563816), OsCRT3 (BAC06263), AtCRT1 (NP_176030), AtCRT2 (NP_172392), and HsCRT (AAB51176) was performed using the ClustalW program at Network Protein Sequence analysis (http://npsa-pbil.ibcp.fr) (39). Aligned sequences were shaded using the BoxShade web server (http://bioweb.pasteur.fr). Residues identical in more than 4 sequences are shaded in red and similar ones are shaded in cyan. Each CRT contains a signal peptide, a globular domain (marked by thick blue lines), a P domain (indicated by a thin gray line), and a C-terminal domain (marked by a red open line). The positions of ebs2 mutations are indicated by blue triangles. Stars denote 2 substitutions in ebs2–2, and diamonds indicate 6 residues thought to be involved in glycan binding. (E) Number of basic and acidic residues in C-termini [the ER retention signal (H/K)DEL was not counted] of AtCRT3, OsCRT3, AtCRT1/2, and HsCRT. The total number of amino acid residues and the net charge of these C-termini are also listed.